Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies have traditionally spanned the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes. In the past year, our studies have focused on human DNA polymerase iota: The cDNA encoding human DNA polymerase iota (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies lead to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase iota (pol iota) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified pol-iota as a 740 amino acid protein. However, the existence of the 740 amino acid pol iota protein has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of pol iota, we were unable to detect the longer 740 amino acid (iota-long) isoform in western blots. However, trace amounts of the iota-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid pol iota. We therefore purified and characterized recombinant full-length (740 amino acid) pol iota-long and compared it to full-length (715 amino acid) pol iota-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between pol iota-long and pol iota-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like pol iota-short, the pol iota-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of pol iota in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform. In 2003, we reported that 129-derived strains of mice carry a naturally occurring nonsense mutation at codon 27 of the Poli gene that would produce a pol iota peptide of just 26 amino acids, rather then the full-length 717 amino acid wild-type polymerase. In support of the genomic analysis, no pol iota protein was detected in testes extracts from 129X1/SvJ mice, where wild-type pol iota is normally highly expressed. The early truncation in pol iota occurs before any structural domains of the polymerase are synthesized and as a consequence, we reasoned that 129-derived strains of mice should be considered as functionally defective in pol iota activity. However, it has recently been reported that during the maturation of the Poli mRNA in 129-derived strains, exon- 2 is sometimes skipped and that an exon-2-less pol protein of 675 amino acids is synthesized that retains catalytic activity in vitro and in vivo. From a structural perspective, we found this idea untenable, given that the amino acids encoded by exon-2 include residues critical for the coordination of the metal ions required for catalysis, as well as the structural integrity of the DNA polymerase. To determine if the exon-2-less pol iota isoform possesses catalytic activity in vitro, we purified a glutathione-tagged full-length exon-2-less (675 amino acid) pol iota protein from baculovirus infected insect cells and compared the activity of the isoform to full-length (717 amino acid) GST-tagged wild-type mouse pol iota in vitro. Reaction conditions were performed under a range of magnesium or manganese concentrations, as well as different template sequence contexts. Wild-type mouse pol iota exhibited robust characteristic properties previously associated with human pol iotas biochemical properties. However, we did not detect any polymerase activity associated with the exon-2-less pol iota enzyme under the same reaction conditions and conclude that exon-2-less pol iota protein is indeed rendered catalytically inactive in vitro.